Physiology & Behavior 127 (2014) 20–26

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Gustatory responsiveness to the 20 proteinogenic amino acids in the spider monkey (Ateles geoffroyi) Jenny Larsson a, Anna Maitz a, Laura Teresa Hernandez Salazar b, Matthias Laska a,⁎ a b

IFM Biology, Linköping University, SE-581 83 Linköping, Sweden Instituto de Neuro-Etologia, Universidad Veracruzana, 91000 Xalapa, Veracruz, Mexico

H I G H L I G H T S • • • •

Spider monkeys clearly prefer the taste of 7 of the 20 proteinogenic amino acids. Spider monkeys clearly reject the taste of 5 of the 20 proteinogenic amino acids. Spider monkeys detect concentrations of amino acids at the millimolar range. The taste responses suggest an evolutionary adaptation to a frugivorous diet.

a r t i c l e

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Article history: Received 9 August 2013 Received in revised form 30 October 2013 Accepted 14 January 2014 Keywords: Gustatory responsiveness Taste preference thresholds Proteinogenic amino acids Spider monkeys Ateles geoffroyi

a b s t r a c t The gustatory responsiveness of four adult spider monkeys to the 20 proteinogenic amino acids was assessed in two-bottle preference tests of brief duration (1 min). We found that Ateles geoffroyi responded with significant preferences for seven amino acids (glycine, L-proline, L-alanine, L-serine, L-glutamic acid, L-aspartic acid, and L-lysine) when presented at a concentration of 100 mM and/or 200 mM and tested against water. At the same concentrations, the animals significantly rejected five amino acids (L-tryptophan, L-tyrosine, L-valine, L-cysteine, and L-isoleucine) and were indifferent to the remaining tastants. Further, the results show that the spider monkeys discriminated concentrations as low as 0.2 mM L-lysine, 2 mM L-glutamic acid, 10 mM L-proline, 20 mM L-valine, 40 mM glycine, L-serine, and L-aspartic acid, and 80 mM L-alanine from the alternative stimulus, with individual animals even scoring lower threshold values. A comparison between the taste qualities of the proteinogenic amino acids as described by humans and the preferences and aversions observed in the spider monkeys suggests a fairly high degree of agreement in the taste quality perception of these tastants between the two species. A comparison between the taste preference thresholds obtained with the spider monkeys and taste detection thresholds reported in human subjects suggests that the taste sensitivity of A. geoffroyi for the amino acids tested here might match that of Homo sapiens. The results support the assumption that the taste responses of spider monkeys to proteinogenic amino acids might reflect an evolutionary adaptation to their frugivorous and thus protein-poor diet. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Amino acids are known to elicit distinct taste qualities in humans, with some of them being described as “sweet” (e.g. glycine), some of them as “bitter” (e.g. L-tryptophan), and some of them evoking complex taste sensations such as “sweet-bitter” (e.g. L-valine) or “meaty-saltybitter” (e.g. L-glutamic acid) [1]. Behavioral data from mice [2,3], rats [4], pigs [5], and musk shrews [6] suggest that these species, too, perceive amino acids as having distinct taste qualities. This should not be surprising given that L-amino acids are the building blocks of proteins and that the ability to detect and discriminate between the tastes of amino acids should therefore be adaptive for animals in order to meet their dietary protein requirements [7]. This notion is supported by studies that ⁎ Corresponding author. Tel.: +46 13 28 1240; fax: +46 13 28 1399. E-mail address: [email protected] (M. Laska). 0031-9384/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.physbeh.2014.01.003

demonstrated that rats rely on their sense of taste to self-regulate their dietary choices when fed an amino acid-imbalanced or -deficient diet [8,9]. In nonhuman primates, the sense of taste has been investigated anatomically [e.g. 10,11], behaviorally [e.g. 12,13], and electrophysiologically [e.g. 14,15] in a number of species. Most studies, however, have so far concentrated on detectability of the five basic taste qualities, usually using sucrose, sodium chloride, hydrochloric acid, quinine hydrochloride, and monosodium glutamate as the only prototypic stimuli, or assessed the properties of artificial tastants such as high-potency sweeteners or taste modifiers [e.g. 16,17]. The few studies that assessed taste responses to amino acids in nonhuman primates usually employed electrophysiological methods [18,19] and found sweet-best and bitterbest fibers to respond to certain amino acids considered “sweet” and “bitter”, respectively, by humans. There is only limited information on taste perception of amino acids in nonhuman primates at the behavioral level [20,21]. Interestingly, the only study so far in which a species of

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nonhuman primates was presented with all 20 proteinogenic amino acids reported that common marmosets (Callithrix jacchus) failed to show a preference for or an aversion to any of these tastants when presented at a concentration of 100 mM and tested against water [20]. Whether this indifference to the taste of proteinogenic amino acids is due to the common marmoset's protein-rich exudativore–insectivore diet [22] or to some other factor remains to be elucidated. The spider monkey (Ateles geoffroyi) is one of the best-studied species of nonhuman primates with regard to taste responses at the behavioral level. It has been found to highly prefer sweet [23–26], salty [27], and umami tastes [27], to be tolerant to sour taste [28], and to reject bitter taste [29]. Spider monkeys are highly frugivorous and thus feed on a diet that is low in protein [30]. Thus, the ability to detect and discriminate between the tastes of amino acids should be particularly important for this primate species. It was therefore the aim of the present study to assess the taste responsiveness of spider monkeys to the 20 proteinogenic amino acids and to determine taste preference thresholds with some of these tastants. To this end, a two-bottle preference test of brief duration (1 min) was employed. This method makes it possible to directly measure preferences for or aversions to tastants and largely rules out the influence of postingestive factors on the animals' ingestive behavior. At the same time, this method allows for a first and conservative approximation of the gustatory sensitivity of the spider monkeys for amino acids.

2. Materials and methods 2.1. Animals Testing was carried out using four adult female spider monkeys (A. geoffroyi). The animals were eight years old at the start of the study. They were maintained at the field station of the Universidad Veracruzana, near the town of Catemaco, in the province of Veracruz, Mexico. The animals were housed as part of social groups in outdoor enclosures with adjacent single cages that could be closed by sliding doors to allow temporary separation of animals for individual testing (for details of maintenance, see [23]). They were fed fresh fruit and vegetables ad libitum. The amount of food offered daily to the animals was such that leftovers were still present on the floor the next morning. Thus, it was unlikely that ravenous appetite affected the animals' ingestive behavior during the tests. As spider monkeys do not normally drink from open sources but meet their water requirements by consuming juicy fruits, no water deprivation schedule was adopted. All animals had participated in previous studies using the same method [29,31] and were completely accustomed to the procedure described below. The experiments reported here comply with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication no. 86–23, revised 1985) and also with current Mexican laws.

2.2. Taste stimuli The following 20 proteinogenic amino acids were used in this study: L-Alanine (L-Ala, CAS# 56-41-7), L-arginine (L-Arg, CAS# 7479-3), L -asparagine ( L -Asn, CAS# 70-47-3), L -aspartic acid (L -Asp, CAS# 56-84-8), L -cysteine ( L -Cys, CAS# 52-90-4), L -glutamic acid (L -Glu, CAS#56-86-0), L -glutamine (L -Gln, CAS# 56-85-9), glycine (Gly, CAS# 56-40-6), L-histidine (L-His, CAS# 71-00-1), L-isoleucine (L-Ile, CAS# 73-32-5), L-leucine (L-Leu, CAS# 61-90-5), L-lysine (L-Lys, CAS# 5687-1), L-methionine (L-Met, CAS# 63-68-3), L-phenylalanine (L-Phe, CAS# 63-91-2), L-proline (L-Pro, CAS# 147-85-3), L-serine (L-Ser, CAS# 5645-1), L-threonine (L-Thr, CAS# 72-19-5), L-tryptophan (L-Trp, CAS# 73-22-3), L-tyrosine (L-Tyr, CAS# 60-18-4) and L-valine (L-Val, CAS# 72-18-4). All substances were obtained from Sigma-Aldrich (St. Louis, MO, USA) and were of the highest available purity.

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2.3. Procedure A two-bottle preference test of short duration was used [32]. The animals were allowed to drink for 1 min from a pair of simultaneously presented graduated cylinders of 100 ml with metal drinking spouts. Tests were performed three times per day, and testing took place in the morning, approximately 2 h before feeding, with 30-minute intervals between tests. 2.3.1. Assessment of taste responsiveness to the 20 proteinogenic amino acids The animals were given a choice between tap water and defined concentrations of the amino acids dissolved in tap water. Two test series were performed: in the first series each amino acid was presented three times (which corresponds to three trials) at a concentration of 100 mM, and in the second series the amino acids were presented three times each at a concentration of 200 mM. During the three trials of a given day, three different amino acids were presented in order to keep up the animals' willingness to cooperate. The position of the stimuli was pseudo-randomized within and between the animals in order to counterbalance possible position preferences. The volume consumed from each bottle was recorded to assess whether the monkeys respond to the given amino acid with a preference for or an aversion to the tastant. 2.3.2. Determination of taste preference thresholds With the seven amino acids that the spider monkeys preferred over water in the previous experiment (glycine, L-proline, L-alanine, L-serine, L-glutamic acid, L-aspartic acid, L-lysine), the animals were given a choice between tap water and defined concentrations of the amino acids dissolved in tap water. With L-valine, an amino acid that the spider monkeys rejected in the previous experiment, we decided to use a 30 mM aqueous sucrose solution rather than water both as the solvent for the amino acid and as the alternative stimulus. This was necessary because spider monkeys cooperate in tests of liquid consumption only as long as at least one of the alternatives is a sapid solution. This concentration of sucrose is only a factor of 10 above the taste preference threshold of the spider monkeys [23] and thus represents a weak sweet stimulus that is unlikely to mask the bitter-tasting L-valine. The same approach has been used successfully in a previous study which assessed the spider monkeys' responsiveness to bitter tastants other than amino acids [29]. With all eight amino acids, testing started at a concentration of 200 mM and proceeded in the following steps (100, 50, 20, 10 mM, etc.) until an animal failed to show a significant preference or aversion. To define the preference thresholds more precisely, this was then followed by intermediate concentrations (e.g. of 30 and 40 mM). To keep up the animals' motivation and willingness to cooperate the testing of the different concentrations did not follow a strict order but was pseudo-randomized. This was true both within a given testing day and between testing days. Each pair of stimuli was presented 10 times per individual animal (which corresponds to a total of 40 trials for the group of four animals, performed during at least four different days). However, if all animals preferred one of the stimuli by more than 80% (relative to the total amount of liquid consumed) after six trials per individual and preferred that same stimulus in at least five out of six trials, testing proceeded with the next concentration. 2.4. Data analysis 2.4.1. Assessment of taste responsiveness to the 20 proteinogenic amino acids The amount of the liquid consumed from each bottle in each trial was recorded for each individual. After the three trials with a given stimulus combination the volumes for each of the two stimuli were summed up for each animal separately and converted to percentages relative to the total amount of liquid consumed from both bottles. Subsequently, for each stimulus combination a mean value (±standard deviation) was built from the percentages of the four individual animals.

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The criteria for a preference at the group level were as follows: the animals were only regarded as significantly preferring one of the two alternative stimuli if they (1) reached the criterion of 66.7% preference (relative to the total amount of liquid consumed) and (2) consumed more from the bottle containing the preferred stimulus in at least 10 out of 12 trials. Accordingly, the animals were regarded as significantly rejecting one of the two stimuli if (1) their preference was less than 33.3% (relative to the total amount of liquid consumed) and (2) they consumed more from the rejected stimulus in not more than 2 out of 12 trials. According to the two-tailed binomial test, the ratios 12/12 and 11/12 as well as the ratios 0/12 and 1/12 correspond to p b 0.01, and the ratios 10/12 and 2/12 correspond to p b 0.05. 2.4.2. Determination of taste preference thresholds The amount of the liquid consumed from each bottle in each trial was recorded for each individual. After the six or ten trials with a given concentration of an amino acid the volumes for each of the two stimuli were summed up for each animal separately and converted to percentages relative to the total amount of liquid consumed from both bottles. Subsequently, for each concentration of an amino acid a mean value (± standard deviation) was built from the percentages of the four individual animals. 66.7% was taken as criterion of preference (i.e., 2/3 of the total amount of liquid consumed). The animals were only regarded as significantly preferring the amino acid over the tap water if they (1) reached 66.7% preference and (2) consumed more from the bottle containing the preferred stimulus in at least eight out of 10, or in five out of six trials per animal, which corresponds to p b 0.05 (two-tailed binomial test). Thus, we defined taste preference threshold as the lowest concentration at which the animals met both criteria mentioned above. Preliminary analysis of the data indicated that there were no reliable differences in choice behavior and liquid consumption between the first, second and third presentation of the day. Intraindividual variability of the amount of liquid consumed across the ten test trials with a given stimulus combination was low and averaged less than 20%. Thus, a theoretically possible bias in the overall preference score due to excessive drinking in aberrant trials did not occur. 3. Results 3.1. Assessment of taste responsiveness to the 20 proteinogenic amino acids Table 1 summarizes the mean performance of the four spider monkeys in the two-bottle preference tests when presented with aqueous dilutions of the proteinogenic amino acids at a concentration of 100 mM and tap water as the alternative stimulus. Results are ranked from the most preferred to the least preferred stimulus. With seven of the 20 amino acids (L-proline, glycine, L-glutamic cid, L-aspartic acid, L-alanine, L-serine, and L-lysine) the animals showed a significant preference whereas one amino acid (L-tryptophan) was significantly rejected. With the remaining 12 amino acids no significant preference or rejection was found, although there were trends in either direction with some of the stimuli. Table 2 summarizes the mean performance of the four spider monkeys in the two-bottle preference tests when presented with aqueous dilutions of the proteinogenic amino acids at a concentration of 200 mM and tap water as the alternative stimulus. Results are ranked from the most preferred to the least preferred stimulus. With three of the 20 amino acids (glycine, L-proline, and L-alanine) the animals showed a significant preference whereas four of them (L-tyrosine, L-valine, L-cysteine, and L-isoleucine) were significantly rejected. With the remaining 13 amino acids no significant preference or rejection was found, although here, too, there were trends in either direction with some of the stimuli. With all 20 amino acids and with both concentrations tested, we observed that, in each trial, each animal sampled both alternatives at least

Table 1 Preference ranking of the 20 proteingenic amino acids presented at a concentration of 100 mM. Amino acid (100 mM)

Mean ± SE

Preference

L-Proline

97.6 ± 0.8 88.9 ± 6.3 86.2 ± 2.9 85.3 ± 3.9 83.4 ± 8.5 71.9 ± 4.3 70.6 ± 7.1 61.7 ± 11.7 58.4 ± 6.5 57.2 ± 23.1 56.5 ± 4.4 54.9 ± 4.6 54.2 ± 4.6 52.2 ± 14.3 50.1 ± 7.1 50.1 ± 8.9 40.5 ± 5.6 38.0 ± 9.2 31.4 ± 11.7 29.3 ± 9.3

12/12 11/12 12/12 11/12 11/12 11/12 11/12 7/12 2/12 8/12 6/12 6/12 6/12 5/12 4/12 4/12 3/12 1/12 5/12 2/12

Glycine L-Glutamic acid L-Aspartic acid L-Alanine L-Serine L-Lysine L-Histidine L-Tyrosine L-Methionine L-Leucine L-Arginine L-Glutamine L-Aspargine L-Valine L-Cysteine L-Threonine L-Isoleucine L-Phenylalanine L-Tryptophan

Bold typeface indicates amino acids that meet both criteria: At least 66.7% preference (or less than 33.3% preference) and at least 10/12 trials with preference (or less than 3/12 trials with preference) for a given amino acid when tested against tap water (two-tailed binomial test, p b 0.05).

once and usually they even tended to switch between bottles more than once. A comparison between the taste preference rankings of the spider monkeys obtained with the amino acids presented at 100 mM and 200 mM, respectively, showed a highly significant correlation between the two (Spearman rs = +0.75, p = 0.0012).

3.2. Determination of taste preference thresholds Fig. 1 shows the mean performance (±SD) of the four spider monkeys when presented with various concentrations of an amino acid and tap water (or, in the case of L-valine, a 30 mM aqueous solution of sucrose) as the alternative. At the group level, the animals significantly discriminated concentrations as low as 80 mM L-alanine, 40 mM glycine, Table 2 Preference ranking of the 20 proteingenic amino acids presented at a concentration of 200 mM. Amino acid (200 mM)

Mean ± SE

Preference

Glycine L-Proline L-Alanine L-Glutamic acid L-Serine L-Leucine L-Lysine L-Aspargine L-Glutamine L-Methionine L-Histidine L-Threonine L-Arginine L-Aspartic acid L-Phenylalanine L-Tryptophan L-Tyrosine L-Valine L-Cysteine L-Isoleucine

96.9 ± 1.2 95.8 ± 2.5 92.5 ± 3.6 83.6 ± 5.8 68.7 ± 8.4 66.2 ± 8.2 64.6 ± 8.2 63.2 ± 7.8 59.0 ± 7.0 50.9 ± 10.5 50.5 ± 17.0 49.4 ± 9.1 47.4 ± 12.1 46.3 ± 4.8 37.7 ± 17.2 37.5 ± 7.2 33.0 ± 11.3 30.2 ± 12.4 28.9 ± 12.7 27.7 ± 6.6

12/12 12/12 12/12 9/12 7/12 8/12 7/12 6/12 4/12 7/12 7/12 7/12 5/12 2/12 4/12 2/12 2/12 2/12 1/12 1/12

Bold typeface indicates amino acids that meet both criteria: At least 66.7% preference (or less than 33.3% preference) and at least 10/12 trials with preference (or less than 3/12 trials with preference) for a given amino acid when tested against tap water (two-tailed binomial test, p b 0.05).

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Fig. 1. Gustatory responsiveness of four spider monkeys to various concentrations of glycine, L-proline, L-alanine, L-serine, L-glutamic acid, L-aspartic acid, L-lysine, and L-valine, respectively, dissolved in tap water and tested against tap water as the alternative stimulus (please note that L-valine was dissolved in a 30 mM sucrose solution and tested against a 30 mM sucrose solution as the alternative stimulus). Each data point represents the mean value (±SD) of ten trials of 1 min per animal. Black data points indicate concentrations at which all animals failed to significantly prefer (or, in the case of L-valine: reject) the corresponding amino acid over water (two-tailed binomial test, p N 0.05).

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L-serine,

and L-aspartic acid, 20 mM L-valine, 10 mM L-proline, 2 mM Lglutamic acid, and 0.2 mM L-lysine from the alternative stimulus. In most cases, interindividual variability of scores was low, as can be inferred from the SDs in Fig. 1. Accordingly, at the individual level, the taste preference threshold values (or, in the case of L-valine, the taste rejection threshold value) for a given tastant maximally differed by a factor of 5 (L-aspartic acid and L-serine), and usually less between subjects. With three of the eight amino acids (L-glutamic acid, L-valine, and L-lysine), all four subjects even scored the same threshold value. With all eight amino acids we observed that, in each trial, each animal sampled both alternatives at least once and, with the exception of the highest concentrations of L-valine, they even tended to switch between bottles more than once. 4. Discussion The results of this study demonstrate that spider monkeys responded with significant preferences for seven of the 20 proteinogenic amino acids when presented at a concentration of 100 mM or 200 mM and tested against water. At the same concentrations, they significantly rejected five of the 20 proteinogenic amino acids. Further, the results show that A. geoffroyi responded to all eight amino acids for which taste preference thresholds were determined at concentrations in the millimolar range, and some of them even at concentrations as low as 0.2 mM (L-lysine) or 2 mM (L-glutamic acid). Although only four animals were tested, the results appear robust as interindividual variability generally was low (see SDs in Tables 1 and 2, and Fig. 1). Further, with all amino acids for which taste preference thresholds were determined, the lowest concentrations presented were consumed at equal amounts compared to the alternative stimulus, suggesting that the preference for (or, in the case of L-valine, the rejection of) higher concentrations of a stimulus was based on taste perception and not on other cues. Although the possibility that olfactory cues might have contributed to the taste preferences and aversions displayed by the spider monkeys cannot be ruled out completely, this appears unlikely for two reasons: firstly, with the exception of the two sulfurcontaining amino acids (L-cysteine and L-methionine), the animals hardly ever sniffed at the spouts of the stimulus bottles during the tests. Secondly, the olfactory sensitivity of spider monkeys for amino acids, substances with a rather low vapor pressure, was found to be in the same millimolar range as found here with their taste sensitivity [33]. Table 3 compares the taste qualities of the 20 proteinogenic amino acids as described by humans [1] and the taste responses obtained with the spider monkeys in the present study. Five of the seven amino acids which the spider monkeys preferred over water (glycine, L -proline, L -alanine, L -serine, and L -lysine) are described as “sweet” by humans although it should be mentioned that all of these five amino acids are, at the same time, described as having a complex taste, that is, they elicit more than one taste quality rather than pure sweetness. Nevertheless, there are only two amino acids which humans describe as “flat to sweet” (L-threonine) or “slightly sweet” (L-valine) for which the spider monkeys failed to display a preference at the concentrations tested. Both of these two amino acids are at the same time described as “bitter” which might explain this finding. This notion is supported by our finding that four of the five amino acids which the spider monkeys rejected (L-tryptophan, L -tyrosine, L -valine, and L -isoleucine) are described as “bitter” by humans (in three of these four cases as the only descriptor suggesting a perception of pure bitterness), and the remaining amino acid (L -cysteine) that was rejected is described as “sulphurous, obnoxious”. Only two among those amino acids which humans describe as “bitter” without additional taste qualities (L-asparagine and L-leucine) were not rejected by the spider monkeys. Taken together, these comparisons suggest a fairly high degree of agreement in the taste quality perception of proteinogenic amino acids between human subjects and spider monkeys.

Table 3 Comparison between the taste qualities of the amino acids as described by humans and the taste responsiveness of the spider monkeys. Amino acids

L-Alanine L-Arginine L-Asparagine L-Aspartic

acid

L-Cysteine L-Glutamic

acid L-Glutamine Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine

Taste qualities as described by humans

Sweet; possibly complex with bitter aftertaste Flat to bitter; alkaline, complex Flat to bitter Flat, sour, slightly bitter Sulphurous, obnoxious Unique, meaty, salty, bitter, sour, complex Flat, sweet, meaty, somewhat unpleasant Sweet, pleasant, smooth, refreshing Flat to bitter, minerally Flat to bitter Flat to bitter (similar to L-isoleucine) Bitter, complex, salty, sweet Flat to bitter; sulphurous, meaty Bitter, possibly complex and strangling Sweet, complex with salty and sour components Flat to sweet; possibly sour, complex Flat to sweet, possibly bitter, sour or fatty Flat to bitter Flat to bitter Flat to bitter, slightly sweet

Taste responsiveness of spider monkeys At 100 mM

At 200 mM

Preferred

Preferred

Preferred Rejected Preferred Preferred

Preferred Rejected

Preferred

Preferred

Preferred

Preferred Rejected Rejected Rejected

Descriptions of taste qualities from [1].

This is not a trivial result considering that the common marmoset (C. jacchus), the only other nonhuman primate species tested so far for its taste responsiveness to all 20 proteinogenic amino acids, failed to display a preference for or an aversion to any of these tastants when presented at a concentration of 100 mM and tested against water [20]. Interestingly, the common marmosets clearly preferred several D-amino acids described as “sweet” by humans over water suggesting that the lack of preferences that they showed with L-amino acids is not due to a general insensitivity of their sense of taste but may rather be due to some other factor. A few other nonhuman primate species have been tested in previous studies for their responsiveness to some of the proteinogenic amino acids and the results show similarities as well as differences when compared to those of the spider monkeys tested here: cynomolgus monkeys (Macaca fascicularis), for example, were found to prefer glycine over water [21]. However, the cynomolgus monkeys failed to show a preference for L-alanine, L-proline, and L-serine which, like glycine, were all preferred by the spider monkeys. The black-handed tamarin (Saguinus midas niger) and the lesser bushbaby (Galago senegalensis) were also found to prefer glycine over water whereas the greater slow loris (Nycticebus coucang) rejected this amino acid [12]. In line with the present findings with spider monkeys, seven species of nonhuman primates, the owl monkey (Aotus trivirgatus), the squirrel monkey (Saimiri sciureus), the common marmoset (C. jacchus), the saddleback tamarin (Saguinus fuscicollis), the black-handed tamarin (S. midas niger), the fat-tailed dwarf lemur (Cheirogaleus medius), and the grivet (Cercopithecus aethiops) were all found to reject L-tryptophan [34]. The few non-primate mammal species that have been tested for their responses to some of the proteinogenic amino acids also show similarities with and differences to those observed in the spider monkeys: pigs (Sus scrofa domestica), for example, were found to prefer glycine, L-alanine, L-asparagine, L-glutamine, L-serine, and L -threonine over water in two-choice preference tests [5]. House musk shrews (Suncus murinus) showed a preference for solutions of glycine, L -alanine, L -valine, L-leucine, L-isoleucine, and L-proline when tested against water and rejected L -cysteine and L -arginine

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Table 4 Comparison of taste preference thresholds in spider monkeys, rats, and mice, and taste detection and recognition thresholds in humans for the eight amino acids tested (in mM).

Human (detect.) Human (recog.) Mice Mice Rats Rats Spider monkeys

Gly

L-Pro

L-Ala

L-Ser

L-Glu

L-Asp

L-Lys

L-Val

Ref.

30.9 25–35 0.1 10 5.8 25 40

15.1 25–27 100 55 19.9 10 10

16.2 12–18 10 18

20.9 25–35

0.06 8–12

0.18 6–8

0.71 80–90

4.16 20–22

[1] [46,47] [2,3] [35] [4] [45]

2

40

0.2

20

0.2 80

79 4.1 40

[6]. Mice (Mus musculus) preferred glycine, L-alanine, L-serine, L-proline, L-threonine, and L-glutamine over water in two-bottle preference tests whereas they rejected L-isoleucine and L-methionine [2,35,36]. Hamsters (Mesocricetus auratus) were indifferent to glycine and rejected L-tryptophan [37], and calves (Bos primigenius) were found to prefer glycine over water [38]. Taken together, these comparisons suggest that certain amino acids such as glycine, L-alanine, and L-proline, which have been described as “sweet” by humans, appear to be preferred by a variety of species from different mammalian orders whereas other amino acids such as L-tryptophan, described as “bitter” by humans, are widely rejected. However, the finding that there are also clear between-species differences in the preferences and aversions displayed towards the taste of the proteinogenic amino acids raises the question as to possible reasons underlying this phenomenon. Previous studies on tastants other than amino acids suggest that differences in dietary specializations may account for the observed differences between species in the tolerance towards substances tasting “bitter” [29,39] or “sour” [28] to humans. Similarly, between-species differences in the preference for substances tasting “sweet” to humans have been found to correlate with differences in dietary specializations [40] and in some cases even the genetic basis of such differences has been elucidated [41–43]. The notion that dietary specialization may also explain differences between species in taste responses to amino acids is supported by our finding that the spider monkeys which are highly frugivorous and thus feed on a diet that is low in protein [30] displayed clear preferences for several proteinogenic amino acids (see Tables 1 and 2) whereas common marmosets which are exudativor-insectivorous and thus feed on a protein-rich diet [22] were found to be indifferent towards all 20 proteinogenic amino acids [20]. Further studies including other species differing in their dietary habits are needed to corroborate this hypothesis. Table 4 compares the taste preference thresholds of the spider monkeys obtained here with those of rats and mice, and with taste detection and recognition thresholds in humans for the eight amino acids tested. Rats (Rattus norvegicus) scored lower threshold values than the spider monkeys with three of the four amino acids tested with this rodent [4,44]. Mice (M. musculus) and spider monkeys outperformed each other with two amino acids each [2,3]. Human taste detection thresholds were lower than the spider monkeys' taste preference thresholds with six of the eight amino acids [1]. However, it should be considered that the sophisticated psychophysical signal detection methods employed with human subjects are likely to be more sensitive than the simple two-bottle preference test used with the spider monkeys. This assumption is supported by findings reporting that taste thresholds obtained with a conditioning paradigm in rhesus monkeys (Macaca mulatta) were up to two log units lower than those obtained using a preference paradigm with the same species [45]. This suggests that the taste sensitivity of the spider monkeys for the amino acids tested here might match that of human subjects. This notion is also supported by the finding that Homo sapiens and A. geoffroyi outperform each other with four amino acids each when comparing the taste preference thresholds of the spider monkeys to

the taste recognition thresholds (rather than the taste detection thresholds) of human subjects (see Table 4) [46,47]. A comparison between the rankings of sensitivity (from highest to lowest threshold values) for the eight amino acids tested with the spider monkeys in the present study and those reported in human subjects [1] shows a positive correlation between the two which falls short of statistical significance (Spearman rs = +0.59, p = 0.12). A comparison between the taste preference thresholds obtained here with those obtained in previous studies with other sets of tastants shows that the sensitivity of the spider monkeys for the amino acids tested falls into the same millimolar range as that for tastants described as “sweet” (sucrose: 3 mM; fructose: 15 mM; glucose: 20 mM [23]) and “sour” (ascorbic acid: 5 mM; citric acid: 5 mM; malic acid: 10 mM [48]), respectively. This suggests that spider monkeys might indeed use not only the relative sweetness and sourness of fruits as gustatory cues for food choice [28] but also the taste of free amino acids. This notion is supported by findings that reported the total abundance of free amino acids in tropical fleshy fruits to be in the millimolar range [49]. Taken together, the results of the present study support the assumption that the taste responses of spider monkeys to proteinogenic amino acids might reflect an evolutionary adaptation to their frugivorous and thus protein-poor diet.

Acknowledgments Financial support by CONACYT Mexico to Laura Teresa Hernandez Salazar (J-51435-IV) is gratefully acknowledged.

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